Mouse fibroblasts growing in vitro are inhibited by glucocorticoids. The cells possess a macromolecule receptor which specifically binds glucocorticoids with high affinity. The research proposal focuses on an examination of the bological and biophysical properties of this receptor macromolecule, and the relationship of the receptor to the phenomenon of steroid hormone resistance. It has been found that glucocorticoid-resistant variants of the fibroblast are grossly deficient in the receptor macromolecule. This project is currently developing information on the intracellular translocations of the receptor macromolecule. The unbound form of the receptor is found in the soluble portion of the cytosol. Upon addition of steroid, binding takes place followed by a temperature- dependent translocation into the nucleus. Within the nucleus, it exists in two forms. The major portion is a steroid-receptor complex which can be readily extracted from the nucleus by 0.3 M KC1 (the "nuclear extractable" form). Energy deprivation (KCN treatment, glucose deprivation) drives most of the nuclear receptor into the residual form. The residual form can be isolated by treating chromatin samples with DNase. The laboratory is currently studying the physicochemical properties (molecular weight, sedimentation rate, axial ratio, isoelectric points) of the three forms of steroid-bound receptor. Available evidence indicates that the receptor macromolecule undergoes a marked allosteric alteration subsequent to binding the glucocorticoid. The change in shape is dependent on temperature and ionic strength of the solvent. We are also preparing spin-labeled analogs of glucocorticoid hormones and hope to examine the receptor-steroid interaction by ESR techniques. Photo affinity labels will also be prepared.